Environmental Health Perspectives Volume 108, Supplement 5, October 2000
Immunohistochemical Localization of Growth Factors and Their Receptors in Uterine Leiomyomas and Matched Myometrium
Darlene Dixon,1 Hong He,1 and Joseph K. Haseman2
1Laboratory of Experimental Pathology, 2Biostatistics Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA
Abstract
Immunolocalization of transforming growth factor alpha (TGF-
), epidermal growth factor (EGF), insulinlike growth factor (IGF)-I, vascular endothelial growth factor (VEGF
165,189,121), basic fibroblast growth factor (FGF)-2, EGF receptor (R), IGF-IRß, and FGFR-1 was studied in uterine leiomyomas and matched myometrial samples taken from seven women (42-47 years of age) in the proliferative phase of the menstrual cycle. Immunolocalization of growth factor peptides was accomplished with either monoclonal or polyclonal antibodies to the amino or carboxy terminus of growth factor peptides or their respective receptors, or against full-length recombinant growth factor. All reactions were conducted using the avidin-biotin complex method. Immunolocalization of TGF-
, EGF, EGF-R, IGF-I, IGF-IRß, FGF-2, FGFR-1, and VEGF was observed in the cytoplasm of smooth-muscle cells of leiomyomas and matched myometrium. The cytoplasm of vascular smooth-muscle cells expressed TGF-
, EGF, EGF-R, IGF-I, IGF-IRß, FGF-2, FGFR-1, and VEGF, whereas the vascular endothelium was positive for TGF-
, EGF, EGF-R, FGF-2, and FGFR-1 in both leiomyomas and matched myometria. Fibroblasts within the fibrous component of some leiomyomas were positive for IGF-I and FGF-2 and minimally positive for FGFR-1. In addition, the extracellular matrix of leiomyomas showed focal localization of FGF-2 and IGF-I in some tumors. When scores of intensity and percent positive staining were compared, IGF-IRß was significantly increased in the leiomyomas compared to matched myometria, whereas EGF was significantly decreased in the uterine leiomyomas compared to matched myometria. In summary, these data revealed growth factors to be expressed differentially in smooth muscle, vascular and fibroblastic cell types of leiomyomas and matched myometria. Specifically, IGF-IRß was significantly increased in leiomyomas; although a similar increase was seen with IGF-I peptide, statistical significance was not achieved. The EGF peptide was significantly decreased in the leiomyomas compared to matched myometrium. These data suggest that IGF-IRß and IGF-I peptide may be one of several growth factor/receptor pathways important in uterine leiomyoma growth during the proliferative phase of the menstrual cycle. In addition, decreased EGF may be secondary to the predominant estrogenic milieu present at time of sampling, as it has been proposed that progesterone, and not estrogen, may regulate EGF.
Key words: growth factors/receptors, immunohistochemistry, leiomyomas, myometrium, uterus. --
Environ Health Perspect 108(suppl 5):795-802 (2000).
http://ehpnet1.niehs.nih.gov/docs/2000/suppl-5/795-802dixon/abstract.html
This article is based on a presentation at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research held 7-8 October 1999 in Research Triangle Park, North Carolina, USA.
Address correspondence to D. Dixon, National Institute of Environmental Health Sciences, PO Box 12233, MDC2-09, Rm. C254, 111 Alexander Dr., Research Triangle Park, NC 27709 USA. Telephone: (919) 541-3814. Fax: (919) 541-2260. E-mail: dixon@niehs.nih.gov
The authors thank G. Flake and A. Nyska for their critical review of this manuscript. The authors also thank N. Flagler for his knowledge and expertise in digital image processing and A. Moore for her help with graphics.
Received 23 February 2000; accepted 12 July 2000.
The role of growth factors in uterine leiomyoma ("fibroids"; myoma) development and growth has not been fully elucidated. It has been suggested that the ovarian steroid hormone responsiveness of fibroids may be mediated, in part, by peptide growth factors that influence the proliferation of smooth muscle, fibroblasts, and the vasculature (
1-3). Genes of growth factors and growth factor receptors may be inducible by estrogen and/or progesterone, thus suggesting that growth factors may serve as paracrine, autocrine, or intracrine mediators of estrogen- and/or progesterone-stimulated growth (
4-6). Uterine leiomyomas may also be the target of environmental chemicals whose biological effects are mediated through the estrogen receptor.
Growth factors such as insulinlike growth factor (IGF)-I, IGF-II, epidermal growth factor (EGF), transforming growth factor-beta (TGF-ß), platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF; FGF-2), vascular endothelial growth factor (VEGF), and their respective receptors have been implicated or hypothesized to play a role in uterine leiomyoma growth (1-3,7-18). Most studies to date have measured the increase or decrease of growth factor expression by mRNA or gene expression studies (8,13,17). Others have conducted receptor assays to assess receptor quantitation (19-22). There are few studies that evaluate and quantitate actual localization of growth factor peptides and receptors in uterine leiomyomas and matched myometrial samples using immunohistochemical techniques (1,2,12,15). In this study, we investigate and quantitate differential immunoexpression of several growth factor peptides and their respective receptors in uterine leiomyomas and matched myometrial samples taken from seven women in the proliferative phase of the menstrual cycle.
Subjects
Tissue from seven women undergoing hysterectomies for symptomatic leiomyomas was included in this study. These women were between 42 and 47 years of age, with a median age of 45.0. All of the subjects had taken no hormonal medication within at least 3 months before hysterectomy. Informed consent was obtained, and the Institutional Review Board of the National Institute of Environmental Health Sciences approved the study.
From three and up to six tumors were collected from each woman. A total of 36 tumors were evaluated for this study. Matched myometrial samples were taken from an area adjacent to but not including the leiomyomas for each woman. All leiomyomas and unaffected myometrial samples were confirmed by histologic evaluation; the endometrium from each uterus was evaluated for phase of the menstrual cycle and found to be weakly proliferative (1/7) to proliferative (6/7). Uterine weights ranged from 201 to 877 g. All patients had multiple fibroids, and tumor sizes sampled ranged from < 2 cm to 5.0 cm in diameter.
Immunohistochemistry
Uterine leiomyoma and myometrial samples were fixed overnight in 10% neutral buffered formalin. Tissues were embedded in paraffin, sectioned at 6 µm and mounted on charged glass slides (ProbeOn Plus, Fisher Scientific, Pittsburgh, PA, USA). Tissues were deparaffinized in xylene and rehydrated with decreasing concentrations of ethanol (100, 95, and 70%). Endogenous peroxidase activity was blocked using 3% H2O2 for 30 min at room temperature. Tissues were trypsinized with a 1:2 dilution of 1
trypsin-EDTA in 1 automation buffer for 10 min at room temperature. Tissues were blocked using normal horse, goat, or rabbit serum at room temperature for 30 min, respective to the secondary antibody used. Tissues were incubated with each of the primary antibodies listed in Table 1 for 24 hr at 4°C at dilutions shown. Negative controls consisted of normal rabbit, goat, or mouse IgG at a concentration the same as the primary antibody. Incubation with respective secondary antibody (rabbit antigoat; goat antirabbit; horse antimouse) was done at room temperature for 60 min. Immunoreactive complexes were detected by avidin-biotin affinity system (Elite Vectastain ABC Kit, Vector Laboratories, Burlingame, CA, USA) and visualized with 3,3´ diaminobenzidine tetrachloride (Sigma Chemical Co., St. Louis, MO, USA) as the chromogen. Tissues were counterstained with Mayer's hematoxylin (Polyscientific Research and Development Corp., Bay Shore, NY, USA) and observed by light microscopy.
A semiquantitative numeric score incorporating overall percent of positive immunohistochemical staining and intensity of immunostaining was determined for each leiomyoma and myometrial sample using the multiplicative quickscore method described by Detre et al. (23). Intensity of staining for each tissue was categorized as negative = 0, weak = 1, moderate = 2, or intense = 3. This number was multiplied by a number generated for the percent of tissue showing positive immunohistochemical staining (0-4% = 1; 5-19% = 2; 20-39% = 3; 40-59% = 4; 60-79% = 5; 80-100% = 6) to generate a quickscore number.
Statistics
For each growth factor/receptor evaluated, quickscores were obtained from a total of 36 tumors and 7 matched myometrial samples. Statistical comparisons were performed on summary of mean quickscores of leiomyomas and the matched myometrial sample quickscore of each patient. The quickscores were compared by the Wilcoxon signed-rank test (24). All comparisons represent the summary of quickscore means of seven patients, with the exception of FGF-R and VEGF, which represent a summary of the quickscore means of three patients (17 tumors; 3 myometrial samples).
Immunohistochemistry
IGF-I, IGF-IRß. A summary of immunohistochemical localization of IGF-I/IGF-IRß and other growth factors/receptors present in uterine leiomyomas and matched myometria is shown in Table 2. For IGF-I peptide, in some of the leiomyoma samples where IGF-I expression was intense, the IGF-I peptide was localized to the connective tissue bands intervening between the smooth-muscle cell bundles of the tumor (Figure 1b). This pattern of staining was not present in the myometrial samples (Figure 1a) or in all of the leiomyomas. In some of the leiomyomas and myometrial samples, there were also focal perivascular regions of epithelioid cells (Figure 1c,d) and fibroblast and epithelioid cell types that were forming perivascular cuffs or infiltrating the intervening connective tissue matrix in the leiomyomas (Figure 1b,d). Minimal to mild focal IGF-I expression was also observed in the vascular, myometrial, and leiomyoma smooth-muscle cells. The expression of IGF-IRß was localized to the cytoplasmic membranes, with some intracytoplasmic staining of smooth-muscle cells of the leiomyomas, myometria, and vasculature (Figure 1e,f). When quickscores were compared, the intensity and percent of tissue staining for IGF-IRß were significantly higher (p < 0.05) in the leiomyomas compared to matched myometrial samples (Figure 2). Although IGF-I expression was increased in some of the tumors because of variability in expression of the peptide from tumor to tumor (and from patient to patient), the mean quickscore for IGF-I was not significantly different from myometrium (Figure 2).
Figure 1. Immunohistochemical localization of IGF-I and IGF-IRß in matched myometrium (a,c,e) and leiomyoma tissue (b,d,f). a,b 88; c,d,e,f 325. IGF-I (a,b,c,d) is localized to the perivascular regions in the myometrium (a,c); however, in the leiomyomas expression is within the connective tissue matrix (b) and more extensive in the perivascular regions (d). Note epithelioid (arrowhead) and fibroblast (arrow) cell types within the extracellular matrix of the leiomyoma (b) and perivascular regions (c,d) of myometrium and leiomyoma samples expressing IGF-I peptide. Inset b: IGF-I-positive fibroblasts (arrows) in the extracellular matrix adjacent to a nest of leiomyoma smooth-muscle cells (smc). 140. IGF-IRß (e,f) is localized to the cytoplasmic membranes (arrows) with some intracytoplasmic staining of myometrial (e) and leiomyoma (f) smooth-muscle cells. Insets c-f: negative control tissue stained with normal goat (c,d) or normal rabbit IgG (e,f).
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Figure 2. Scoring of immunohistochemical localization of IGF-I and IGF-IRß in leiomyomas and matched myometrial samples. The data are the averaged mean ± SD of 36 leiomyomas and 7 myometrial samples. *p < 0.05.
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TGF-, EGF, EGF-R. TGF- was primarily detected in the cytoplasm of the vascular smooth-muscle cells and vascular endothelium (Figure 3a,b) of the leiomyomas and myometria. The myometrial and leiomyoma smooth-muscle cells showed minimal to no expression of TGF-. There was no statistically significant difference in mean quickscores of leiomyomas and myometria for TGF- expression (Figure 4). The peptide EGF was expressed in the cytoplasm of myometrial, leiomyoma, and vascular smooth-muscle cells, and in the cytoplasm of the vascular endothelium (Figure 3c,d). Similar EGF-R localization patterns were observed in the leiomyomas and matched myometria, with staining being associated with the cytoplasmic membranes along with intracytoplasmic staining (Figure 3e,f). In some but not all of the tumors, EGF-R expression was decreased. EGF peptide was significantly decreased in the leiomyomas compared to the myometrial samples. EGF-R showed a similar reduction; however, it did not achieve statistical significance (Figure 4).
Figure 3. Immunohistochemical localization of TGF-, EGF, and EGF-R in matched myometrium (a,c,e) and leiomyoma (b,d,f) tissue. 325. TGF- is localized to the cytoplasm of vascular smooth-muscle cells with minimal staining of the myometrial (a) and leiomyoma (b) smooth-muscle cells (smc), and vascular endothelium (arrows). Note expression of EGF in the cytoplasm of myometrial (c) and leiomyoma (d) smooth-muscle cells and staining of vascular endothelium (arrow) in the myometium (c). Note EGF-R expression in the cytoplasm of vascular, myometrial (e), and leiomyoma (f) smooth-muscle cells. Insets a-f: negative control tissue stained with normal mouse (a,b) or normal rabbit (c-f) IgG.
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Figure 4. Scoring of immunohistochemical localization of TGF-, EGF, and EGF-R in leiomyomas and matched myometrial samples. The data are the averaged mean ± SD of 36 leiomyomas and 7 myometrial samples. *p < 0.05.
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FGF-2, FGF-RI (Flg). FGF-2 was localized to the cytoplasm of smooth-muscle cells of the vasculature, myometrium, and leiomyomas (Figure 5a,b). Fibroblasts within the connective tissue stroma of leiomyomas showed cytoplasmic staining for FGF-2 (Figure 5b). FGF-2 was also localized to the cytoplasm of vascular endothelial cells. Depending on the degree of fibrogenesis, varying amounts of FGF-2 were observed in the extracellular matrix of some of the leiomyomas and within the cytoplasm of the fibroblasts in these regions. Expression of FGFR-1 was localized to the cytoplasmic membrane and scantily present throughout the cytoplasm of myometrial, vascular, and leiomyoma smooth-muscle cells, and fibroblasts (Figure 5c,d). The vascular endothelium was positive for FGFR-1 in leiomyoma and myometrial samples (Figure 5c). In this study there was no difference in mean quickscores of FGF-2 and FGFR-I in leiomyomas and matched myometrial samples (Figure 6).
Figure 5. Immunohistochemical localization of FGF-2, FGFR-1, and VEGF in matched myometrium (a,c,e) and leiomyoma (b,d,f) tissue. 325. Note immunoexpression of FGF-2 in smooth-muscle cells of the myometrium (a) and leiomyoma (b). Note positive staining of fibroblasts (arrows) in intercellular matrix of leiomyoma (b). Also note staining of smooth muscle and endothelium of blood vessels (arrowheads) in the myometrium (a) and leiomyoma (b). FGFR-1 showed cytoplasmic and membranous staining of myometrial (c) and leiomyoma (d) smooth-muscle cells. Also note staining of endothelium and smooth-muscle cells of blood vessels (arrows) from matched myometrial (c) sample. Note VEGF expression in smooth-muscle cells of the myometrium (e) and leiomyoma (f), and staining of vascular smooth-muscle cells (arrow) shown in the leiomyoma (f). Insets a-f: negative control tissue stained with normal rabbit (a-d) or normal goat (e,f) IgG.
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Figure 6. Scoring of immunohistochemical localization of FGF-2 and FGFR-1 in leiomyomas and matched myometrial samples. The data are the averaged mean ± SD of 36 leiomyomas and 7 myometrial samples (FGF-2), or 17 leiomyomas and 3 myometrial samples (FGFR-1).
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VEGF. Immunolocalization of VEGF was within the cytoplasm of smooth-muscle cells of the vasculature, myometrium, and leiomyomas (Figure 5e,f). There was minimal expression of VEGF in the cytoplasm of the vascular endothelial cells. There was no statistically significant difference of quickscores for this growth factor in the myometrial and leiomyoma samples examined (Figure 7).
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Figure 7. Scoring of immunohistochemical localization of VEGF in leiomyomas and matched myometrial samples. The data are the averaged mean ± SD of 17 leiomyomas and 3 myometrial samples.
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The exact role of growth factor peptides and their receptors in the growth and development of uterine leiomyomas is unknown. It is known that these tumors are responsive to ovarian hormones, namely progesterone and estrogen, and these ovarian hormones have been found to regulate the secretion of growth factor peptides and the expression of their receptors (
4-6,14). A mechanism has been proposed for uterine leiomyomas in which estrogen- and/or progesterone-related growth effects observed in these tumors may be indeed secondary to the autocrine/paracrine/intracrine effects of overexpression of growth factor peptides or upregulation of receptor expression by cell types comprising uterine leiomyomas.
The seven women in this study were all in the proliferative phase of the menstrual cycle, which would suggest that the major ovarian hormonal influence at the time of sampling was estrogen. Under the conditions of this study, we found that there was markedly increased expression of IGF-IRß in the leiomyomas. IGF-I peptide expression was also increased in some tumors; however, this effect was not seen in all patients, and as a result the mean quickscore for all tumors was not statistically significant from matched myometria. In those tumors where there was intense positive staining for IGF-I peptide, the staining extended from perivascular staining of epithelioid cells to extensive perivascular localization of IGF-I-positive epithelioid and fibroblast cell types, with involvement of the bands of connective tissue separating the smooth muscle tumor cells. The IGF-I-positive epithelioid cells described in our study may possibly represent IGF-I-positive inflammatory cells of the monocyte/macrophage cell series previously described in human myometrium (25), although specific markers for these cell types would be necessary for definitive identification. Van Der Ven et al. (10) showed a 2.4 times higher concentration of IGF-I in uterine leiomyomas compared to myometrial samples. When values were corrected for the contribution of intravascular IGF-I to the total IGF-I concentration in leiomyomas and myometrial samples, the difference was 3 times higher. This increase in IGF-I peptide in leiomyomas, as shown in in vitro studies, was due to increased IGF-I uptake in leiomyoma smooth-muscle cells rather than synthesis. Our data support these findings, in that we observed only minimal to moderate focal immunolocalization of IGF-I peptide in myometrial and leiomyoma smooth-muscle cells; however, in the epithelioid and fibroblast cell types within the perivascular and extracellular matrix regions of some leiomyomas, there was intense expression of IGF-I peptide. This suggests a possible paracrine mechanism for IGF-I-induced growth in leiomyomas. The increased synthesis of IGF-I by nonleiomyoma smooth muscle cell types, coupled with increased expression of IGF-IR as we observed in this study, could possibly explain the increased growth observed in some tumors. However, factors such as IGF-binding proteins and their effect upon IGF-I binding and bioavailability must be considered (26). Similar to our finding of increased expression of IGF-IRß in leiomyomas compared to matched myometrial samples, other studies have shown increased concentrations of type 1 IGF receptor in uterine leiomyomas compared to myometrium (11,21).
The decrease in EGF immunoexpression observed in this study may be secondary to the predominant ovarian estradiol secretion of the women at the time of sampling. Harrison-Woolrych et al. (13) reported that EGF production in uterine leiomyomas is cyclic, with EGF mRNA production in untreated leiomyomas being increased during the secretory phase of the cycle compared to EGF mRNA from myometrium of the normal uterus. Although these authors found no difference in the amount of EGF mRNA in leiomyomas and normal myometrial samples during the proliferative phase of the cycle, these data and our findings of significantly decreased EGF peptide expression in the leiomyomas challenge the hypothesis that EGF synthesis in leiomyomas is mediated by estrogen and question the role of progesterone in regulating EGF production and uterine leiomyoma growth (13,14). Others have found no difference in EGF mRNA levels between leiomyomas and matched myometrial samples (27). In the case of the EGF-R, we found no statistically significant difference between mean quickscores of leiomyomas and matched myometrium, although the mean quickscore was lower than the matched myometrial samples. Hofmann et al. (20) have reported no difference in cyclical variation of the EGF-R in normal myometrium or leiomyomas or any difference in binding between the two tissues. Conversely, Tommola et al. (21) have shown that leiomyoma tissue binds less EGF than samples of matched myometrium, and attributes this decrease in binding to changes in receptor concentration versus receptor affinity. In culture, leiomyoma cells have been reported to have fewer EGF-R sites/cell than matching cultures of myometrial cells (19).
There was no difference in immunolocalization of TGF- in uterine leiomyomas and matched myometrial samples in this study. Localization was primarily within the cytoplasm of perivascular smooth-muscle cells and endothelium, with minimal to no expression in the smooth-muscle cells of leiomyomas or myometrium. In mice, we have found that TGF- is not expressed in the smooth-muscle cells of uterine leiomyomas; however, this growth factor is expressed in the smooth-muscle cells of uterine leiomyosarcomas in mice (28). TGF- has been implicated in the transformation of normal fibroblasts into a malignant phenotype (29). These data suggest that overexpression of TGF- may be important in malignant transformation of mesenchymal cell types.
Immunoexpression of FGF-2 (bFGF) and FGF-R in the uterine leiomyoma and myometrial samples was not different. Although a few of the leiomyomas showed minimal to moderate FGF-2 localization in the extracellular matrix, the overall intensity and percent of tissue staining in the leiomyomas compared to myometrial samples was not statistically different. This may be due, in part, to variation in the amount of the extracellular matrix present in each tumor, with some tumors having less with minimal to no FGF-2 localization in these regions, and others having moderate to expansive amounts of connective tissue and moderate FGF-2 localization. Mangrulkar et al. (2) found elevated bFGF mRNA transcripts in uterine leiomyomas, and immunohistochemically the peptide appeared localized primarily within the extracellular matrix. These authors proposed that enhanced growth of leiomyomas might be related to the large quantities of bFGF observed in the extracellular matrix. Others have observed strong FGF-2 immunoreactivity of smooth-muscle cells in normal myometrial samples with no staining of fibroblasts or extracellular matrix (30). FGF-2 is mitogenic for leiomyoma and myometrial cells; however, leiomyoma cells appear to be less responsive (31). Although we found no difference in FGFR-1 expression, others have reported FGFR-1 expression in the myometrium and leiomyomas throughout the menstrual cycle, with immunoreactivity for the receptor stronger in the myometrium than in leiomyomas during the proliferative phase of the cycle (1,3).
Although quantitation of the angiogenic growth factor VEGF was not statistically different between matched myometrial and leiomyoma samples, the quickscore values were consistently higher in uterine leiomyomas compared to myometrial samples. Harrison-Woolrych et al. (17) have reported no differences in VEGF mRNA levels in untreated leiomyomas compared to normal myometrium. These authors also found no significant difference in VEGF mRNA levels in the proliferative and secretory phases of the cycle in leiomyomas, in contrast to the normal myometrium in which these authors have shown VEGF mRNA levels to be significantly higher in the secretory phase of the cycle.
In summary, we speculate that differential expression of growth factor peptides and their receptors by cell types in uterine leiomyomas is regulated by ovarian hormones, in part, and by some local autonomous factor(s) within a given tumor that triggers growth factor synthesis and/or receptor upregulation and progression of cells through the cell cycle. We propose that different growth factors, growth factor receptors, and signaling pathways are coordinately turned on and off throughout the menstrual cycle and throughout the life span of the tumors, and that one growth factor/receptor pathway alone is not solely responsible for the growth of these tumors. This has been shown with IGF-I and myometrial smooth-muscle cells, in that IGF-I alone is a weak mitogen for uterine smooth-muscle cells; however, in combination with EGF and PDGF-BB there is significant smooth muscle cell growth (25). By understanding the role of growth factors, their receptors and signaling pathways, and the interaction of growth factors and ovarian hormones in uterine leiomyoma growth, intervention strategies might be devised that would allow perturbation of these biological pathways and disruption of uterine leiomyoma growth. Futhermore, defining the role of genetic susceptibility and the contribution of environmental estrogenic and/or endocrine-disrupting chemicals to the development and progression of uterine leiomyomas will be critical to understanding the basic biological mechanism(s) of this disease process.
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Last Updated: October 3, 2000
